Wnt/Beta-Catenin signaling is involved in the carcinogenesis of different solid malignant tumors. The interaction of Creb-binding protein (CBP) with Beta-Catenin is a pivotal component of the Wnt/Beta-Catenin signaling pathway. The first aim of this study was to evaluate the association of CBP expression with survival in patients with human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC). Second, the in vitro effects of the inhibition of CBP/Beta-Catenin interaction were analyzed. In particular, the effects of ICG-001, an inhibitor of CBP/Beta-Catenin interaction, on proliferation, cell death, modulation of Wnt/Beta-Catenin target expression, and cell migration were examined in vitro. High CBP expression is significantly associated with better survival on mRNA and protein levels. Furthermore, we observed cytotoxic as well as anti-migratory effects of ICG-001. These effects were particularly more potent in the HPV-positive than in the -negative cell line. Mechanistically, ICG-001 treatment induced apoptosis and led to a downregulation of CBP, c-MYC, and Cyclin D1 in HPV-positive cells, indicating inhibition of Wnt/Beta-Catenin signaling. In conclusion, high CBP expression is observed in HPV-positive HNSCC patients with a good prognosis, and ICG-001 showed a promising antineoplastic potential, particularly in HPV-positive HNSCC cells. Therefore, ICG-001 may potentially become an essential component of treatment de-escalation regimens for HPV-positive HNSCC. Further studies are warranted for additional assessment of the mechanistic background of our in vitro findings. Head and neck squamous cell carcinoma (HNSCC) derives from the mucosa of the larynx, oropharynx, hypopharynx, nasal cavity, and paranasal sinuses and nasopharynx. They account for almost half a million new cases annually and represent the sixth most common malignancy worldwide . HNSCC iThe role of double-stranded HPV in HNSCC, and OPSCC in particular, has been well known for the last two decades . HPV is In general, HPV-positive OPSCC is linked to excellent survival outcomes. Nevertheless, therapy for HPV-positive OPSCC is still very intense. To date, cisplatin is the most extensively used systemic agent in first-line therapeutic approaches for this type of cancer. Although cisplatin is very effective as systemic therapy, it is associated with multiple severe side effects which include, among others, acute kidney failure, hearing loss, and neuropathy .In HPV-positive OPSCC, treatment de-escalation options are currently heavily discussed . HoweverThe Wnt/Beta-Catenin (WBC) pathway is an important, well-conserved signaling pathway in cancer. It is responsible for different processes during embryonic development, such as proliferation, migration, and differentiation . FurtherBello et al.  hypothes33H32N4O4 [The small molecule inhibitor “ICG-001” blocks the interaction between Beta-Catenin and the Creb-binding protein (CBP), therefore inhibiting the WBC pathway . Chemica3H32N4O4 . Specifi3H32N4O4 . Several3H32N4O4 , nasopha3H32N4O4 , gastric3H32N4O4  and, pan3H32N4O4 .However, to the best of our knowledge, no studies have analyzed its effects in HPV-positive malignancies yet. Therefore, our study aimed to assess the association of WBC signaling with survival in HPV-positive HNSCC. In particular, we aimed to assess the prognostic potential of CBP expression in silico. Furthermore, using in vitro experiments, we sought to analyze the antineoplastic effects of CBP/Beta-Catenin inhibition using a small molecule inhibitor ICG-001 in an HPV-positive cell line as well as HPV-negative cell line serving as a control.Crebbp mRNA expression.The data for a total of 41 patients with HPV-positive HNSCC were retrieved from “The Cancer Genome Atlas” (TCGA). Crebbp mRNA expression is associated with OS, the log-rank test was performed and showed significantly longer OS in the high expression group . Crebbp mRNA expression.The median overall survival (OS) for the whole cohort was 1.70 years (range 0.36–4.75 years). In order to test if A total of 29 patients fulfilled the inclusion criteria and had available follow-up data. The detailed patient and tumor characteristics are presented in p = 0.016 and p = 0.039, respectively). The median OS and disease-free survival (DFS) for the whole cohort were 1.8 years (range 0.3–12.3 years) and 1.5 years (range 0.0–9.8 years), respectively. Analog to the TCGA analysis, significantly longer survival times were observed in the group with high CBP expression as calculated by the OTV and a minimum patient distribution of 20% per group  in combination with irradiation (from 0–8 Grey (Gy)) in order to assess the cytotoxicity and a potential synergistic effect in combination treatment. The cell viability was measured 72 h post-treatment using a resazurin assay. A dose-dependent proliferation inhibition could be observed in both cell lines, and the ICWe could generate evidence that ICG-001 treatment leads to a downregulation of CBP A,B. In aA significant change in the amount of early apoptotic, late apoptotic, and dead cells could be observed in one or both tested cell lines post 72 h treatment with ICG-001 at 1.5 µM and 5 µM, respectively. In particular, the early apoptotic cell percentage increased significantly in Cal27 cells upon treatment . Moreover, downregulation of WBC transcriptional targets was observed in ICG-001-treated SCC154 cells, further supporting the hypothesis of HPV-induced deregulation of the WBC pathway.The current study revolved around the role of the WBC pathway in HPV-positive HNSCC in terms of its prognostic relevance and therapeutic potential of its inhibition. First, we provided evidence that high Crebbp mRNA and protein expression with survival in HPV-positive HNSCC in silico. High CBP expression was linked to better survival times. Similar results on protein level were shown by Rühlmann et al. [Crebbp) could be the result of a higher viral load and have a similar positive prognostic effect, such as the high viral load, in those tumors. Indeed, further studies are needed in order to decipher the exact mechanistic background behind this observation.The interaction between CBP and Beta-Catenin is an important component of the WBC pathway . Therefon et al.  for patin et al. . Higher n et al.  were ablThe above-noted interaction of the HPV with the WBC pathway via the HPV-associated oncoproteins E6 and E7 was further elucidated in a recent study by Muñoz-Bello et al. . They weIn the study by Schultz et al. , the exp2/day for 1 week. Indeed, the cytotoxicity rate was acceptable, with only one patient developing hyperbilirubinemia [neurofibromin 2 gene mutated meningioma, using in vitro and in vivo xenograft models.The various side-effects of cisplatin, including acute kidney failure or hearing loss , are welubinemia . Ko et aubinemia  conducteubinemia  reportedAs noted, the majority of patients with HPV-positive OPSCC associate with good outcomes. Therefore, efforts are made to investigate possible treatment de-escalation regimens ,37. ThesCrebbp expression and better survival outcome was observed in silico and validated on protein level in an independent cohort. For future validation studies of our in vitro data, additional HPV-negative and HPV-positive cell lines should be included. In addition, the current study did not provide information on the exact mechanistic effects of ICG-001 treatment in vitro, particularly for HPV-negative cells. Despite the above-mentioned shortcomings of the study, CBP expression may become a valuable survival biomarker for HPV-positive HNSCC. Moreover, the ICG-001 may become a candidate for targeted therapy in HPV-associated HNSCC. Taken together, we showed that CBP expression might be a valuable prognostic marker and the specific blockade of its interaction with Beta-Catenin a potential therapy option for low-risk patients with HPV-positive HNSCC.Limitations of the current study include that the size of the TCGA cohort is relatively small. Nevertheless, a significant association between high Crebbp) with OS in HPV-positive HNSCC was analyzed. The data were retrieved using the TCGA database  [https://portal.gdc.cancer.gov/legacy-archive/search/f. accessed on 30 December 2021) and normalized utilizing the R package “TCGA biolinks” (version 2.19.0) and processed in R . Survival analysis was performed with packages “survival” and “survminer”  in R. Kaplan–Meier curves were plotted with “ggplot” (version 3.3.3). The OTV of Crebbp expression in regard to OS was calculated (>OTV was considered as high expression) with a patient distribution of at least 20% per group. The whole cohort was stratified according to the OTV into high and low and a Kaplan–Meier survival curve for OS was calculated and plotted.First, the association of CBP expression on mRNA level (gene name of CBP: ch 2021) . All patAll patients with a histologically verified HPV-positive HNSCC, primarily surgically treated in our institution from 1 January 2012 to 31 December 2019, were included in this study. Patients with recurrent disease or a second malignancy were excluded from the analysis. Furthermore, data on age, sex, tumor staging, and other clinically relevant data were retrieved. The association of CBP expression on protein level with OS and DFS was analyzed.For this analysis, a TMA of formalin-fixed paraffin-embedded tumor samples of all included patients was constructed. This was performed with the computer-assisted tissue microarray platform . The subsequent staining was performed on 4 µm slices.2O2 and Ultra V Block were used in order to block the endogenous peroxidase activity. Then, the samples were incubated for 1 h (h) with the primary antibody against CBP 1:100  at room temperature. Subsequently, the primary antibody enhancer and horseradish peroxidase enhancer were applied for 10 and 15 min, respectively. In order to visualize the staining, the UltraVision Plus Detection System DAB Plus Substrate System  was utilized. The counterstaining of the tissues was performed with hematoxylin Gill II . The tissues were scanned using the Panoramic Flash 250 II. The expression of the CBP was analyzed. We performed the quantification of the staining using QuPath (Version 0.2.3). Tissue was analyzed for the percentage (0–100%) of stained cells. The survival analysis was conducted with the R packages “survival” and “survminer” . The optimized threshold values in regards to overall and DFS were calculated, serving as a cutoff for the patient stratification into high and low CBP groups (>optimized threshold was considered as high). The calculation of the optimized threshold was performed with a patient distribution of at least 20% per group. Kaplan Meier curves were plotted with “ggplot”.The immunohistochemical staining was performed with the Lab Vision UltraVision Kit , with colon tissue serving as a positive control. For the staining, the TMA was first dewaxed and dehydrated. Then, antigen retrieval was performed using ethylenediaminetetraacetic acid in a microwave (10 min (min) at 600 Watt). Next, 3% H2  and cultivated in DMEM supplemented with 10% FBS and 1% P/S. Cells were split once per week by trypsinization. The inhibitor ICG-001 was acquired from Selleckchem . All in vitro experiments were performed at least three times. Mean values ± standard deviations (SD) were calculated and used for further analysis and graphical representation. Displayed graphical presentation show representative images.Next, we evaluated the effect of WBC pathway inhibition in vitro. For these experiments, we used the HPV-positive HNSCC cell line SCC154. The Cal27, an HPV-negative HNSCC, cell line served as a control. They were both acquired from the American Type Culture Collection  and were regularly tested for mycoplasma contamination. Dulbecco’s modified eagle’s medium (DMEM), Penicillin/Streptomycin (P/S) and fetal calf serum (FBS) were obtained from Gibco . Cells were kept in a humidified environment at 37 °C and 5% COhttps://synergyfinder.org, accessed on 31 January 2021) was used for calculating synergistic/additive effects of irradiation and ICG-001 treatment.In order to analyze cell proliferation, cells were seeded in 96-well plates  in the following fashion: SCC154 20,000 cells/100 µL/well; Cal27 5000 cells/100 µL/well. A set of five replicates for each dose was used. One day after seeding, cells were treated with 100 µL of inhibitor diluted in DMEM. Ascending inhibitor concentrations were used and ranged between 0.625 and 10 µM. Dimethyl sulfoxide (DMSO)  treated cells served as control. Furthermore, cells were irradiated at 2, 4, and 8 Gy . The Synergyfinder tool , diluted in DMEM, was added to each well, and the cells were incubated. The measurements were performed with a TECAN Spark reader ; for Cal27 after 90 min of incubation and for SCC154 after 150 min.50 inhibitor values calculated in the cytotoxicity assay, corresponding inhibitor concentration ranges of ICG-001 were used in subsequent experiments for two cell lines.Based on ICIn order to assess the anti-migratory effects of WBC inhibition in a dose-dependent manner, the cells were seeded in 24-well plates  with 150,000 Cal27 cells/well and 700,000 SCC154 cells/well. The medium was prepared with DMEM containing 1% P/S and 1% FBS . Cells were seeded at 100% confluency, and simultaneously, a migration gap was created by a culture insert  which was removed 24 h after seeding. Then, the cells were washed with 1x DPBS  once followed by treatment with ICG-001. Concentrations of 1.5 and 6 µM in Cal27 and 0.5 and 1 µM in SCC154 of ICG-001 were used.The initial visualization was performed immediately after treatment with a TECAN Spark reader. The same was performed after 24 and 48 h in SCC154 and after 18 h in Cal27 . Before visualization, cells were washed with 1x DPBS, and fresh DMEM was added (in order to remove dead and floating cells). The analysis and calculation of the gap closure percentage were performed with ImageJ 1.53e .50 concentrations of ICG-001 . After 72 h of treatment, the cells were trypsinized, washed, and pelleted, and the apoptotic cell death was determined by Annexin V /7-AAD  co-staining, following the instructions from the manufacturer. Data from stained cells were acquired with a BD FACSCanto II . Analysis of results was performed with the FACSDiva software 8.0.1 .In order to assess cell apoptosis after treatment with ICG-001, a FACS Annexin V assay was performed. Firstly, 80,000 Cal27 and 150,000 SCC154 cells were seeded into each well on a 12-well plate. On the next day, the cells were either treated with DMSO (vehicle control) or the approximate ICAfter trypsinization, washing, and pelleting of the cells, the cell pellet was lysed in RIPA buffer  supplemented with phosphatase  and protease inhibitors . For all samples, equivalent amounts of protein were diluted in RIPA buffer and separated by a 10% SDS-PAGE. Next, proteins were transferred to a nitrocellulose membrane  with a Trans-Blot Turbo Transfer System . In the following step, membranes were blocked with 5% BSA in 1 × TBS/0.1% Tween-20 for 1 h and incubated overnight at 4 °C on a roller shaker with the following primary antibody: β-TUBULIN , PARP , CBP , c-MYC  and Cyclin D1 . On the following day, membranes were incubated with the second antibody (specific for the host species of the primary antibody) at a dilution of 1:5000 (in 1× TBS / 0.1% Tween-20) for 1 h at room temperature. The target protein expression was visualized using the ECL™ Prime Western Blot System from Cytiva  and the ChemiDoc XRS+ . Quantification of results from immunoblotting was conducted using ImageJ 1.53e .The statistical analysis of the patient cohort data was performed with the R software. The level of statistical significance was set at 0.05, two-tailed. Survival analysis was performed using R, as described above, and the log-rank test was used to assess if the two groups have a statistically significant difference in survival times.www.graphpad.com. accessed on 5 December 2020).In order to assess the differences in gap closure between different inhibitor concentrations in the migration assay and to analyze results from the Annexin V/7-AAD assay, the two-way ANOVA test was used. Differences in protein expression between treated and untreated cells were assessed using the one-way ANOVA test. Graphical presentation of cell culture results, as well as the one-way/two-way ANOVA analyses, were performed with GraphPad Prism . The antineoplastic potential seems to be higher in SCC154, supporting the hypothesis of HPV-induced WBC pathway deregulation. Indeed, a strong downregulation of WBC transcriptional targets was observed in the HPV-positive cell line SCC154 treated with ICG-001. Furthermore, high CBP expression could possibly facilitate the identification of low-risk patients with HPV-positive HNSCC. Finally, ICG-001 may become a viable therapy option for these low-risk patients, particularly as a component of proposed treatment de-escalation regimens for this patient group. Indeed, further research is warranted in order to decipher the exact molecular effects of ICG-001, as well as to assess further in vitro and in vivo antineoplastic effects of ICG-001, particularly for HPV-positive HNSCC.


The primary objective of this study was to assess whether 18F-NaF activity can assess the presence and predict the progression of CT detectable vascular calcification.Microcalcifications cannot be identified with the present resolution of CT; however, 18F-NaF PET-CT at baseline and after 6 months or 1-year follow-up were used. The target to background ratio (TBR) was measured on PET at baseline and CT calcification was quantified in the femoral arteries at baseline and follow-up. 128 patients were included. A higher TBR at baseline was associated with higher calcification mass at baseline and calcification progression . In areas without calcification at baseline and where calcification developed at follow-up, the TBR was .11–.13 (P < .001) higher compared to areas where no calcification developed.The data of two longitudinal studies in which patients received a 18F-NaF is related to the amount of calcification and calcification progression. In areas where calcification formation occurred, the TBR was slightly but significantly higher.The activity of The online version of this article (10.1007/s12350-020-02031-5) contains supplementary material, which is available to authorized users. However, the availability of data on 18F-NaF PET is limited and validation studies are challenging.Atherosclerosis is a major cause of disability and death worldwide.18F-NaF uptake is associated with cardiovascular risk factors.18F-NaF binds to the surface of hydroxyapatite crystals. Subsequently, the hydroxyl ion is exchanged with the 18fluoride ion on the surface of hydroxyapatite to form fluorapatite. Vascular 18F-NaF uptake might therefore be a measure of the calcification surface area and able to visualize smaller calcifications than CT as these microcalcifications have a large surface area relative to the amount of calcium.18F-NaF specifically binds areas of microcalcification, and therefore can identify areas of active calcification.18F-NaF activity is associated with incident and progressive vascular calcification on CT in the femoral arteries, which is an arterial bed where motion artifacts do not play a major role.Higher 18F-NaF PET-CT at baseline and after 1 year (TEMP) or 6 months (VITACAL) follow-up were used. In both studies, TBR on PET and calcium mass on CT were measured at baseline and follow-up.For this study, data from two longitudinal medication studies  in which patients underwent an 18F-NaF PET-CT was performed. The study was previously described in detail.11The Treatment of Ectopic Mineralization in Pseudoxanthoma Elasticum Trial (TEMP) was a single center, randomized, double-blind, placebo-controlled trial conducted in the University Medical Center Utrecht in the Netherlands. Patients with a clinical diagnosis of Pseudoxanthoma Elasticum (PXE),18F-NaF PET-CT was performed. The study was previously described in detail.12The VItamin K and vascular calcification (VITACAL) study was a single center, randomized, double-blind, placebo-controlled trial conducted in the University Medical Center Utrecht in the Netherlands. Patients diagnosed with diabetes mellitus type 2, aged ≥ 40 with presence of arterial disease  were included. Patients were randomized between 6 months treatment with 360 microgram vitamin K (menaquinone-7) daily and placebo. At baseline and after 6 months treatment a full body The CT and PET acquisition parameters were described previously.max) was determined by manually drawing a circular region of interest (ROI) around the femoral artery on every other slice . The mean SUVbackground was determined by using the average of three ROIs drawn in the superior vena cava at the level of the aortic arch on consecutive slices. The target to background ratio (TBR) was calculated by dividing the SUVmax by the SUVbackground. The observer was blinded for treatment status and clinical information.On PET the standardized uptake value .Statistical analysis was performed with RStudio version 1.1.414 . Data are provided as mean ± standard deviation in case of a normal distribution and as median [interquartiles] if non-normally distributed. Categorical variables are provided as A multilevel linear model was used since data were clustered per patient and per side. The natural log transformation of the TBR (as measured at baseline) was used as dependent variable, adjusted for age, gender, BMI and renal function (creatinine level). Subsequently, stratified analyses per treatment group were performed. Presented are the results after back transformation.Subgroup analysis was performed in slices without calcium at baseline. Slices in which calcium progression occurred were compared to slices without calcium at follow-up.Finally, we investigated whether high TBR values were associated with calcium progression. This was only investigated in the placebo group, since treatment might interfere with calcium progression. From all slices without calcium at baseline, the slice with the highest TBR at baseline was selected in each patient in each lower limb. This was defined as the ‘hottest slice’. If multiple slices had the same highest TBR, they were all marked as the hottest slice. The calcium mass at follow-up on these hottest slices was compared to the remaining slices using the Mann–Whitney-U test.P = .67). Baseline characteristics are provided in Table In total 74 patients were included. One patient did not receive the follow-up scan and was therefore excluded. After the consensus meeting seven lower limbs in six different patients  were excluded because the baseline and follow-up data could not be matched sufficiently. Ultimately, 72 patients (139 lower limbs) were included with 6,294 slices in total. Forty-one percent  of the CT slices showed calcium at baseline. Of those slices, 1,108 slices (43%) showed calcium progression at follow-up. The baseline TBR of those slices was 3.2 compared to 3.1 in slices without calcium progression  and the amount of calcium at baseline . Analysis stratified per group (placebo or etidronate) showed the same direction and effect sizes (Online Appendix 4).Results of univariate analysis are provided in Online Appendix 3. Multivariate analysis Table  showed tβ = 1.131 [1.105-1.159], P < .001).There were 3,691 slices without calcium at baseline. In 8% (286 slices) of those slices, calcium developed at follow-up. Figure P < .001). The hottest slice (148 slices) showed a mean calcium mass score at follow-up of 3.204 (SD 8.684) vs 1.064 (SD 4.728) in the 1,525 other slices (P < .001). In 20% (30/148) of the hottest slices and in 8%  of the remaining slices calcium developed at follow-up. Figure The hottest slice analysis in slices without calcium at baseline in the placebo group showed that the progression of the calcium mass at follow-up was significantly higher for the hottest slice . Baseline characteristics are provided in Table In total 68 patients were included in the VITACAL study. Eight patients did not receive the follow-up scan and were therefore excluded. After the consensus meeting 18 lower limbs in 14 different patients  were excluded. Therefore 56 patients (101 lower limbs) were included with 4,642 slices in total. Forty-eight percent  of the slices showed calcium at baseline. Of those slices, 1,152 slices (25%) showed calcium progression at follow-up. The baseline TBR of those slices was 2.7 compared to 2.6 in slices without calcium progression  and the amount of calcium at baseline . Analysis stratified per group (placebo or vitamin K) showed the same direction and effect sizes (supplement 4).Multivariate analysis Table  showed tβ = 1.113 [1.084-1.142], P < .001).There were 2,430 slices without calcium at baseline. In 13% (322 slices) of those slices, calcium developed at follow-up. Figure P = .960). In 3/32 (9%) of the hottest slices and in 130/1,321 (10%) of the remaining slices calcium developed at follow-up. Figure The hottest slice analysis in slices without calcium at baseline in the placebo group showed that the progression of the calcium mass at follow-up was not significantly different for the hottest slice (slice with the highest TBR at baseline). The hottest slice (32 slices) showed a mean calcium mass score at follow-up of .304 (SD 1.098) vs .345 (SD 1.097) in the 1,321 other slices  and therefore the clinical relevance might be limited.In this longitudinal study, 18F-NaF activity and disease progression are limited and involve small patient groups. Jenkins et al.18F-NaF activity was strongly correlated to both the amount of calcium at baseline and calcium progression. However, 18F-NaF activity was not able to independently predict clinical outcomes . Ishiwata et al.18F-NaF active sites) with a large range in follow-up. The aorta and the common iliac artery were studied. There was no correlation between 18F-NaF activity and baseline calcium; however, the difference in calcium volume and Agatston calcium score were associated with a higher 18F-NaF activity. No correction for potential confounders was performed. De novo calcification occurred in 19/96 (20%) of the hotspots and was related to a longer follow-up interval of on average 5 years, but was not related to a higher TBR at baseline. In the current study de novo calcifications occurred in 20% (TEMP) and 9% (VITACAL) of the hotspots.Longitudinal studies which assess the relationship between 18F-NaF adsorption. Tissue fluoride was only found in the presence of calcification and microcalcifications showed greater levels of fluoride compared to macrocalcifications. 18F-NaF did only bind to the surface of the micro- and macrocalcifications. Recently, Creager et al.18F-NaF binds to the surface of both micro- and macrocalcifications. 18F-NaF is a marker of active calcification which is more likely to occur in the early stages of plaque formation, and not a specific marker of microcalcification. This explains the relationship between the amount of calcification and 18F-NaF activity, which was also reported in several earlier studies.16It would be very promising if a non-invasive biomarker could distinguish between microcalcifications in high-risk plaques and macrocalcifications in relatively stable plaques. Ex vivo research was performed by Irkle et al.18F-NaF activity. These might represent areas with microcalcifications, not yet detectable by CT. In both studies we found that the hottest slice at baseline had a higher calcification mass at follow-up. In the literature, several different methods are used to define hotspots. Hotspots can be defined visually, using a fixed cut-off or using a study or patient specific cut-off as was performed in the current study. Since 18F-NaF activity is dependent on many variables such as age, BMI, renal function, injected dose and PET technology,Of special interest are areas without calcification, as measured on CT, but with increased 18F-NaF activity might allow identification of microcalcifications. Fourth, the coronary arteries, carotid bifurcation and aorta are vascular beds in which atherosclerosis is dominant, but in the femoral artery also medial arterial calcifications occur.25This study has several limitations. First, both studies were clinical trials evaluating a bisphosphonate and vitamin K, respectively. Since the TBR was defined at baseline, it is not possible that this was influenced by treatment. However, the delta calcium can be influenced since the TEMP trial showed that bisphosphonate treatment reduced the amount of arterial calcification.18F-NaF activity in the femoral arteries is associated with the amount of CT calcification and CT calcification progression. Areas in which CT calcification occurred at follow-up, showed a slightly higher TBR at baseline, although the differences were small. This supports the concept that PET can detect calcifications below the resolution of CT to some extent, which may be of interest for studies in persons who do not have CT detectable calcifications.In conclusion this is the first longitudinal study showing that 18F-NaF activity in the femoral arteries is related to the amount of calcification and calcification progression. To some extent, PET-CT can detect calcifications below the resolution of CT, and identify locations where calcifications are likely to develop on CT. This may be of interest for intervention studies in the early phases of the calcification process.This study showed that Online Appendix 1: Plots of relationship between TBR at baseline and the calcium mass at baseline and follow-up for the TEMP study (PDF 6962 kb)Online Appendix 2: Plots of relationship between TBR at baseline and the calcium mass at baseline and follow-up for the VITACAL study (PDF 5181 kb)Online Appendix 3: Results of univariate linear regression (DOCX 14 kb)Online Appendix 4: Results of multilevel linear regression models stratified per treatment group for both studies (DOCX 20 kb)Supplementary material 5 (M4A 5455 kb)Supplementary material 6 (PPTX 284 kb)Below is the link to the electronic supplementary material.


Cervical disc prostheses are used to preserve motion after discectomy, but they should also provide a near-physiological qualitative motion pattern. Nevertheless, they come in many completely different biomechanical concepts. This caused us to perform an in-vivo MR-based biomechanical study to further investigate cervical spine motion with the aim to gain new information for improving the design of future cervical arthroplasty devices.Fifteen healthy volunteers underwent MRI-investigation (in order to avoid radiation exposure) of their cervical spines from C3 to C7; for each segment centers of rotation (COR) for flexion / extension were determined from 5 different positions, and CORs for lateral bending from 3 different positions. The motion path of the COR is then described and illustrated in relation to the respective COR for maximum flexion / extension or lateral bending, respectively, and the findings are translated into implications for a better biomechanical prosthesis-design.The COR for flexion / extension does not remain constant during motion. The CORs for the respective motion intervals were always found at different positions than the COR for maximum flexion /extension showing that the COR moves both along the x- and the y-axis throughout flexion / extension. For lateral bending a completely independent COR was found above disc-level.Flexion / extension is not a simple circular motion. Disc prostheses need a variable COR for flexion / extension below disc level with the capability to move both along the x- and the y-axis during motion, plus a second completely independent COR for lateral bending above disc level to closely replicate in-vivo motion. These findings are important for improving the biomechanical design of such devices in the future. Biomechanical studies investigating cervical spine motion and describing how the cervical vertebral bodies exactly move against each other in healthy subjects are essential for the design of cervical disc prostheses.A large variety of such prostheses are – or have previously been - available on the market for preserving motion after discectomy. However, they significantly differ in design and position of the COR. Some have one fixed COR below the motion segment , others a COR above the motion segment level with capability for additional sagittal translation , again others a variable COR  or no articulating surfaces at all but an elastic nucleus . An analysis of the motion pattern after insertion of a Bryan-, a Prestige STLP or a Discover prosthesis in patients after cervical discectomy could show that a more flexible biomechanical design can contribute to a better physiological motion .The great variety of biomechanical studies available include in vitro and in vivo studies using different techniques like plain flexion-extension radiographs –5, biplaFifteen healthy volunteers  with no previous symptoms of cervical spondylosis underwent MRI-investigation of their cervical spines after giving informed consent to the study protocol which was approved by the Ethic commission of the Medical University of Vienna (EK Nr. 571/2007).All investigations were done using a 1.5 T MRI .o. T2-weighted median-sagittal slices showing the entire contours of the vertebral bodies C3 to C7 were used for biomechanical calculations. This protocol allowed acquisition of 5 different data sets during maximum flexion and extension and therefore enables the determination of 4 different intermediate CORs for each motion step . In addition to these intermediate CORs, also for the entire motion between ME and MF a total COR can be calculated. The x / y-coordinates of the intermediate CORs are compared with the x / y-coordinates of the COR for total flexion / extension, and the motion path of the COR is illustrated in relation to the COR for total flexion / extension.Flexion / extension was recorded in 5 different positions for each volunteer. Data for flexion / extension were collected from the following positions: Maximum extension (ME), intermediate extension (IE), neutral position (N), intermediate flexion (IF) and maximum flexion (MF). The volunteers were asked to actively move their heads into these positions and were then supported with cushions to remain in the respective position during MRI data acquisition. The cushions were used to have similar positions of the head for all volunteers only. The motion-amplitudes between these 5 different positions were approximately 3o. The volunteers were asked to actively move their heads to maximum left- and right-bending and were supported with cushions to remain in the respective position. T2-weighted frontal MRI-sections were collected showing the entire contours of the vertebral bodies C3 to C7 for biomechanical analysis. The respective z / y-coordinates of the CORs were analyzed for detection of significant changes during lateral-bending.Lateral-bending was recorded in 3 different positions, therefore receiving data from 2 separate motion amplitudes of approximately 4The coordinate-system for motion analysis of flexion / extension was determined using a line through the most superior anterior and the most superior posterior point of the respective vertebral body. The cutting point with a second line through the most posterior inferior and the most posterior superior point of the respective vertebral body was defined as the center of the coordinate-system with the x-axis passing through the most superior anterior point of the respective vertebral body, the y-axis directing cranially rectangular to the x-axis, and the z-axis rising orthogonally against the viewer Fig. a. For laFigure To capture the coordinates of the reference points A and B the software AutoCAD  was used. Data processing from MRI pictures to AutoCAD software was done by one single investigator; this required marking of the 4 corner-points of the respective vertebral bodies on the MRI pictures in order to create a quadrangle covering the vertebral bodies to allow better overlay of the respective vertebral bodies and to use all 4 edge-points of the quadrangle for a more precise calculation of the COR. Coordinate-calculation was done using Microsoft Excel software . In flexion / extension both the COR from maximum extension to maximum flexion was calculated as well as 4 more CORs from the respective motion-steps in between. In lateral-bending, the COR from maximum left- and right-bending was calculated as well as 2 more CORs from the respective steps between left-to-neutral and right-to-neutral-bending.The t-test was used for determination of significance regarding the differences between the respective data-sets with a significance level of α = 0.05.o (SD 12.7). Table o (SD 9.9). Table Fifteen datasets were analyzed; the mean ROM for flexion / extension from C3 to C7 was 53.4o SD 2.7. TablThe following coordinates  were found for flexion / extension: C3/4: x4.8 / y-5.8 (2.3 / 5.6); C4/5: x4.8 / y-3.8 (2.3 / 3.7); C5/6: x4.8 / y-4.0 (2.8 / 3.1); C6/7: x4.9 / y-1.1 (3.2 / 2.6). Figure In all 15 volunteers, the respective CORs for the 4 separate flexion/extension-intervals were calculated and compared with the coordinates for the respective CORs for maximum flexion / extension. The CORs for ME-IE, IE-N, N-IF and IF-MF are summarized in Table In all investigated levels the respective COR did not remain constant during flexion / extension but changed its coordinates.Level C3/4: from ME-IE there is no significant difference for the x- and the y-coordinates ; from IE-N a significant difference was found for the x-coordinates (x: p = 0.005) but not for the y-coordinates (y: p = 0.337); from N-IF a near-significant difference for the x-coordinates was found (x: p = 0.066) but not for the y-coordinates (y: p = 0.187); from IF-MF no significant difference was found both for the x- and y-coordinates . Figure Level C4/5: from ME-IE, no significant difference both for the x- and the y-coordinates was detected ; from IE-N a significant difference was found for the x-coordinates (x: p = 0.049) but not for the y-coordinates (y: p = 0.842); for N-IF a significant difference for x- and a near-significant difference for the y-coordinates was detected ; the final interval from IF-MF reveals a non-significant change both for the x- and the y-coordinates . Figure Level C5/6: From ME-IE the x-coordinates differ significantly (x: p = 0.026) but not the y-coordinates (y: p = 0.792); from IE-N no significant difference was found both for the x- and y-coordinates ; from N-IF, significant differences were detected both for the x- and for the y-coordinates ; for the final interval from IF-MF no significant differences could be found . Figure Level C6/7: No significant differences for the x- or y-coordinates were found for the ME-IE-interval ; from IE-N the x-coordinates did not change significantly (x: p = 0.074), however a significant difference for the y-coordinates was detected (y: p = 0.033); for the N-IF-interval a non-significant difference was found both for the x- and y-coordinates ; for the final interval IF-MF no significant changes for x- or y-coordinates were detected . Figure This data demonstrates that flexion / extension in the lower cervical spine cannot be described as a simple orbit defined by a constant COR. From C3/4 to C5/6, a similar motion-path for the COR during flexion / extension can be identified: During the ME-IE, the IE-N and also the N-IF intervals the COR migrates caudally and posteriorly. During the IF-MF interval the COR moves again cranially and anteriorly. The change of the y-coordinates for the respective CORs demonstrates that from C3/4 to C5/6 the upper vertebral body of the respective motion segment rotates with a smaller radius at the final motion parts during a flexion / extension maneuver than during the intermediate part of this motion. The reasons why the coordinates for the C6/7-COR migrate in a different manner than the CORs from C3/4 to C5/6 however cannot be clearly answered with this data and requires further investigation.The following coordinates  were found for maximum lateral bending: C3/4: z-7.0 / y8.1 (3.2 / 7.3); C4/5: z-5.9 / y14.9 (3.8 / 6.4); C5/6: z-6.1 / y15.9 (2.9 / 9.3); C6/7: z-9.1 / y9.8 (8.6 / 9.2). Other than in flexion / extension, the COR for lateral bending is located within the upper vertebral body of the respective motion segment ; C4/5: z-8.0 / y10.5 (5.3 / 6.8); C5/6: z-5.8 / y13.4 (7.2 / 7.9); C6/7: z-4.4 / y9.4 (13.6 / 9.9). Table Our data shows that in order to resemble natural motion, cervical arthroplasty devices need 2 separate and independent CORs for flexion / extension and lateral-bending. The COR for flexion / extension should be located below disc level and should be able to change its position during motion both along the x- and the y-axes, so it fits the different locations of the CORs at the respective segments, it allows smaller and larger radii, and it allows radius change during motion to facilitate tilting at the final parts of flexion / extension. Traveling of the COR along the x-axis allows translation in combination with rotation.The separate COR for lateral-bending should be located above disc level, it need not be variable, but it must allow rotation through an oblique sagittal axis (ascending from anterior to posterior) in order to facilitate coupled motion of lateral-bending with rotation.Figure This study showed that the COR for flexion / extension is located below disc level, but not at a stationary position and changes its position over the motion period. Flexion / extension is not a circular motion but is facilitated through a completely variable COR. For lateral bending a separate COR was found above disc level. This COR remains constant throughout motion and is located at an oblique sagittal axis, therefore allowing coupled lateral bending and rotation.Cervical disc prostheses are used to preserve motion after discectomy. It is generally accepted that they should preferably resemble physiological motion as close as possible. Nevertheless, they come in a great variety of completely different biomechanical concepts. At least at the beginning, the basic idea was to use ball-socket designs understanding that flexion / extension is a circular motion guided by the curvature of the facet joints. But even if the surface of the facet joints roughly appears spherical and therefore might determine a circular motion , these jA considerable number of studies investigated the ROM of the cervical spine Tables  and 2 2, 25, 27.Less information is found in the literature regarding the COR. Studies published by Penning, Amevo and van Mameren , 18, 19 Frobin described flexion / extension as a combined rotation and translation , but stiVan Mameren presented a biomechanical analysis based on data derived from a cineradiographic study: 25 radiographic frames were taken during flexion / extension and separately analyzed . HoweverAnderst and Baillargeon contributed in their studies to a better understanding on 3D-motion of the cervical spine during flexion / extension, lateral bending and rotation –22; FlexFor lateral bending and rotation Anderst precisely described coupled motion in his 3D-analysis. However, this study shows that lateral bending and rotation are not two separate complex 3D-motions, but they rather appear as one relatively constant rotation around a sagittal oblique axis. The steeper the angle of this axis, the higher is the rotational component, a flat angle - as it is found in the lower cervical motion segments – causes a higher bending component than rotational component . TherefoOur study demonstrates that flexion / extension is not a simple circular motion following an orbit defined by one single constant COR, but the radius for the rotational component in flexion / extension varies within the respective motion segments during motion in addition to the already known segment-dependent decrease from C3/4 down to C6/7.Figures For the COR in lateral-bending we found no significant changes for the respective CORs during this type of motion. We suppose that the uncovertebral joints together with the facet joints function as a more rigid guidance for lateral bending and therefore keep the COR more constant than in flexion / extension.The major limitation of this study is the small number of volunteers. However, many other studies about biomechanics in cervical arthroplasty published in the literature have similar small cohorts –36. It iWe used MRI for data acquisition and manual digitizing for biomechanical calculation. We are aware that there are more precise techniques, using biplanar radiography plus high resolution CT, for instance, resulting in sub-millimeter precision . HoweverFor data processing from MRI pictures to AutoCAD software, marking of the 4 corners of the vertebra was performed by a single person. Intra-rater and inter-rater variability of these markings were not determined, which can lead to variability in the COR locations. However, we expect that pooling of the data from the 15 subjects will reduce this variability.We are also aware that our coordinates were calculated in millimeters and not as a percentage of the respective vertebral body dimension and therefore do not take into account the individual differences of the size of the volunteers‘different vertebral bodies. As shown in Table The ROM in our study is in the lower range of previously published data referring to functional x-ray, but even very sophisticated other studies  did not Even if we cannot provide coordinates in sub-millimeter precision, we believe the data received from our study is still sufficiently valid to conclude how the biomechanical design of disc prostheses can be further improved.In summary, our study showed that in flexion / extension the CORs of the investigated intermediate flexion / extension-intervals differ from the COR of the respective maximum flexion / extension for all levels from C3/4 to C6/7. Thus, the study showed that the COR is located below disc level, but not at a stationary position and changes its position over the motion period. Comparing with literature and our findings, flexion / extension is not a simple circular motion. For lateral bending a separate COR was found above disc level. This COR remains constant throughout motion and is located at an oblique sagittal axis, therefore allowing coupled lateral bending and rotation. We believe these findings can influence the design of cervical disc prostheses in future. Simple ball-socket design does not allow physiological motion; however, even simple 2-piece devices – as shown in our work – can replicate physiological motion provided the gliding partners are designed according to the biomechanical findings we presented.In order to more closely replicate in-vivo motion arthroplasty devices should have 2 independent CORs, one on either side of the disc level: For flexion / extension a variable COR below disc level that enables the upper vertebral body to rotate with flexible radii together with translation during the middle part and tilting during the end part of flexion / extension. For lateral bending the COR should be above disc level, and lateral-bending should be facilitated through an oblique sagittal axis therefore allowing coupled ipsilateral rotation, and the angle of this axis should be variable for the respective segments increasing from approx. 20 degrees for C6/7 to approx. 40 degrees for C3/4.


Soil and sediment microorganisms are highly phylogenetically diverse but are currently largely under-represented in public molecular databases. Their functional characterization by means of metaproteomics is usually performed using metagenomic sequences acquired for the same sample. However, such hugely diverse metagenomic datasets are difficult to assemble; in parallel, theoretical proteomes from isolates available in generic databases are of high quality. Both these factors advocate for the use of theoretical proteomes in metaproteomics interpretation pipelines. Here, we examined a number of database construction strategies with a view to increasing the outputs of metaproteomics studies performed on soil samples.The number of peptide-spectrum matches was found to be of comparable magnitude when using public or sample-specific metagenomics-derived databases. However, numbers were significantly increased when a combination of both types of information was used in a two-step cascaded search. Our data also indicate that the functional annotation of the metaproteomics dataset can be maximized by using a combination of both types of databases.A two-step strategy combining sample-specific metagenome database and public databases such as the non-redundant NCBI database and a massive soil gene catalog allows maximizing the metaproteomic interpretation both in terms of ratio of assigned spectra and retrieval of function-derived information.Video abstractThe online version contains supplementary material available at 10.1186/s40168-021-01139-1. Soil hosts complex microbial ecosystems which are crucial for numerous ecosystem services, including plant growth and animal life . These eMetaproteomics involves protein extraction and trypsin proteolysis, detection of the resulting peptides by tandem mass spectrometry, interpretation of MS/MS spectra to assign peptide identities, and higher-level interpretation in terms of taxonomy and function , 49. MS/Soil/sediment metaproteomics is currently challenging because a large proportion of organisms in soil samples have yet to be taxonomically characterized  and onlyDifferent databases built from metagenomics data acquired on a sediment sample were evaluated for metaproteomics based on the number of PSMs as main criterion. For this, a soil core was collected from the Seine River floodplain at the Bouafles site (France) located downstream of Paris. The 1-m core was cut up into 3-cm slices. A shotgun metagenome sequencing dataset comprising ~ 87 million Illumina paired-end reads was acquired for the slices corresponding to 17–28-cm depth in the soil core after extracting DNA from a pool of the five corresponding slices. Figure p-value thresholds  were tested for the first-round search to estimate the impact of this parameter on the final results. The second search was performed at decoy-free 1% FDR. Figure X-axis represents the size of the databases used in the final step of the cascaded search. Notably, for all conditions tested, the result variability estimated on the three experimental metaproteomic datasets was quite low, at less than 0.5% in most cases. The one-step search method allowed between 2.2 and 6.5% of MS/MS spectra to be assigned, with the maximum reached using the PLASS database. The sixgill database search performed poorly (only 2.2% MS/MS spectra assigned) even though it was the smallest, and theoretically the best-adapted to the proteomic data format. The two-step database search method significantly increased the proportion of MS/MS spectra assigned, with 3-fold higher values recorded for most conditions. Although this increase was expected, the results reveal that the improvement ratio depends strongly on the stringency of protein selection during the first identification round. Here, optimal p-values could clearly be identified for each database: 0.10 for sixgill, 0.20 for MGH-6RF and PLASS, and 0.30 for MGH-FGS and FGS. Using the two-step search method, higher numbers of confident PSMs were assigned, reaching at best 7.3% for sixgill, 13.0% for MGH-FGS, 14.1% for MGH-6RF, 17.3% for PLASS, and 20.5% for FGS. As with the one-step search, the two-step search strategy performed better with the FGS and PLASS databases, but a clear advantage was noted for the FGS database. Unexpectedly, among the sequencing-read-assembly strategies, a better attribution rate was obtained for PLASS compared to MEGAHIT. This result highlights the power and reliability of a strategy based on assembly of peptide sequences rather than nucleic acid sequences and demonstrates the added value of retaining variants that are discarded by the MEGAHIT algorithm. These results also show that predicting coding sequences after assembly (MGH-FGS) does not provide significant advantages over six-frame translation (MGH-6RF) in the two-step search method, as these databases allowed 13.0% and 14.1% MS/MS assignment, respectively. This result directly contrasted with that of the one-step search strategy, where 6.1% and 4.5% of MS/MS spectra were assigned, respectively. In conclusion, the highest attribution rate (20.5%) and coverage of the microbial metaproteome was obtained with the FGS database when queries were performed at p-value 0.30 in the first search round, using a large database with only short sequences .Proteins were extracted from three equal aliquots of the same section of the soil sample (slice 20–23 cm of the sampled soil core). The peptides derived from the biological triplicates after trypsin proteolysis were analyzed by nanoLC-MS/MS, producing 59,501, 59,917, and 59,141 MS/MS spectra. These three datasets were subsequently used separately to estimate search variability across the different databases even if the biological samples taken for metagenomics and metaproteomics do not match perfectly. Figure p-value thresholds, SGC performed better than NCBInr, with 19.5% versus 15.5% of MS/MS spectra assigned to peptide sequences. Because the two databases could be complementary in terms of environmental sequence coverage, we also assessed the effect of merging SGC and NCBI sub-databases at various p-values (SGC+NCBI). As indicated in Fig. p-value threshold was 0.30. Interestingly, this assignment ratio was higher than that obtained with the classical approach, consisting in nucleic acid sequence assembly and protein sequence prediction (MGH-FGS). These results suggest that, in the future, generic databases — which are continuously expanding to include new environmental metagenomics projects and NCBInr updates — could perform as well as sample-specific metagenomics databases, even when treating difficult environmental samples. This prospect would decrease the per-sample cost of metaproteomics analyses.As shown in Fig. p-value of 0.30). Table We next went on to test the effect of a combination of sample-specific metagenomics databases and generic databases on the attribution rate for MS/MS spectra. The best-performing reduced databases from the two-step search strategy were selected: FGS_0.30, PLASS_0.30, MGH-6RF_0.30, NCBI_0.30, and SGC_0.30 . Through the list of proteins it provides, and their abundances, metaproteomics brings a new dimension to the study of microbiota by delivering the list of organisms present in a sample and their respective biomasses , and by To construct the most appropriate database for use when interpreting metaproteomics data, it is generally recommended to use metagenomics data acquired for the same sample. From the sampled soil, ~ 87 million Illumina paired-end reads were recorded, corresponding to 13 Gbp of sequenced nucleotides. Whether this sequencing effort comprehensively represents the microbial community found in the sample is a key question. In some soil studies, the cumulated efforts made to analyze a large collection of samples is considerably greater. For example, a total of 730 Gbp of sequenced nucleotides were obtained for the analysis of soil communities in phosphorus-deficient and phosphorus-rich tropical soils . SimilarThe metagenomics reads obtained in this study were treated either with MEGAHIT, FragGeneScan, or sixgill. Our results using the five constructed databases confirmed that a strategy with two query rounds, as recommended for unusually large databases , performMicrobacterium oleivorans A9 strain [With the datasets considered here, the best PSM attribution rate was obtained for the FragGeneScan CDSs predicted directly from trimmed reads (20.5%). As expected, this attribution rate was lower than those obtained with similar instruments when studying single organisms for which a well-annotated genome is available. For example, a rate of 61% PSM assignment was reported for the 9 strain . However9 strain ;. The coThe high quality of theoretical proteomes from isolates available in generic databases such as NCBInr and their large numbers advocate for use of these resources in metaproteomics interpretation pipelines. Indeed, the use of selected annotated genomes has previously been explored , 50, 75,Previous studies indicated that merging protein sequence databases from several samples might improve the peptide identification rate , 62. HerTwo significant criteria to consider when assessing the power of metaproteomics is how many of the peptides/proteins identified have taxonomy- and function-derived annotations. In metaproteomics, the taxonomical annotation is commonly performed with taxon-specific peptides using the lowest common ancestor approach, such as with the Unipept tool . HoweverIn conclusion, combining sample-specific metagenomics data and generic databases in a two-step database search performed best for the soil sample analyzed in the present study, both in terms of ratio of assigned spectra and retrieval of function-derived information. Amalgaming a massive soil gene catalog and the generalist NCBInr database resulted in almost the same outcome. This result opens up broad prospects for the application of metaproteomics to soil samples, which includes a highly challenging matrix, as well as broad microbial diversity, and extensive complexity.A soil core was sampled on May 23 2018 from a floodplain at Bouafles near the Seine River (France). The site has already been well characterized in terms of sedimentation and chemicals , 41. TheSoil DNA was extracted and sequenced by GenoScreen  from 1 g of lyophilized sample using an optimized protocol . BrieflyHomo sapiens sequences. FragGeneScan v1.3 [Paired-reads were processed using the MEGAHIT workflow into the ASaiM Galaxy framework . They wecan v1.3  was applhttp://vm-lux.embl.de/~hildebra/Soil_gene_cat/ (accessed on 22 March 2021). NCBnr was downloaded from https://www.ncbi.nlm.nih.gov/ on 3 January 2018).The soil gene catalog  was downg. The resulting pellet was resuspended in 40 μL LDS 1X (Invitrogen) containing 5% beta-mercaptoethanol, sonicated for 5 min in an ultra sound bath and then heated to 99 °C for 5 min. Soluble proteins (25 μL per well) were subjected to SDS-PAGE gel electrophoresis on NuPAGE 4–12% Bis-Tris gel (Invitrogen) for 5 min at 200 V in MES/SDS 1X running buffer (Invitrogen). Proteins were stained for 15 min with Coomassie SimplyBlue SafeStain (Thermo Fisher Scientific), and then in-gel proteolyzed with trypsin gold (Promega) for 1 h at 50 °C, as recommended [The proteins from 5 g of soil were extracted using the NoviPure Soil Protein Extraction Kit (Mo-Bio) as recommended by the supplier. After centrifugation, proteins from the 10-ml supernatant were precipitated by adding 2.5 ml trichloroacetic acid (50% w/v). Proteins were collected by centrifugation for 10 min at 6000×ommended .−1 using a 90-min gradient of mobile phase A (0.1% HCOOH/100% H2O) and phase B (0.1% HCOOH/80% CH3CN). The gradient used was developed from 4 to 25% B in 70 min and then from 25 to 40% B in 20 min. The mass spectrometer was operated in Top20 data-dependent acquisition mode. Full MS scans were acquired from 350 to 1800 m/z at a resolution of 60,000 and the 20 most abundant precursor ions were sequentially selected for fragmentation with a dynamic exclusion time of 10 s. The resolution for the fragment scans was 15,000. Only ions with 2 or 3 positive charges were selected for fragmentation. MS/MS spectra were interpreted using Mascot Daemon software  indicating 5-ppm tolerance for the parent ion and 0.02-Da tolerance for secondary fragments, 2+ and 3+ as possible peptide charges, a maximum of two missed cleavages, carbamidomethylation of cysteine as fixed modification, oxidation of methionine as variable modification, and trypsin as proteolytic enzyme. The FDR threshold was set at 0.01 using a decoy-free FDR method based on a mixture-model of four beta distributions which has been shown well adapted for handling large proteogenomics and metaproteomics datasets and databases [p-value thresholds  for the first search round to select the protein sequences. The most time-consuming search (13 h) was noted for the first step NCBI database interrogation.Peptides were analyzed on a Q-Exactive HF mass spectrometer (Thermo) coupled to an Ultimate 3000 nano LC system (Thermo), as described previously . Trypticatabases . The twoe-value threshold 10, and percentage identity above 50%. The GOSlim terms  associated with the Uniprot accession number were retrieved for each protein. KEGG annotation was performed using the GhostKOALA [Functional annotation of identified proteins was based on sequence similarity searches carried out with Diamond BLASTP (v0.8.22.84)  against ostKOALA  web servostKOALA  desktop


Breast cancer is one of the most difficult malignancies to treat. Therapeutics is used to target and kill the cancer cells. Non-human oncolytic viruses have the ability to cause cell death directly to cancers. The objective here was to investigate the role of Vesicular Stomatitis Virus (VSV) Matrix (M) protein in autophagy in the breast cancer cell line.  Two different VSV wild type and mutant (M51R) M protein constructs were produced. Breast cancer cell line BT-20 was transfected by either wild type or mutant vectors. Transfection efficiency was measured using a fluorescent microscopy. Expression of VSV M protein was investigated at protein level. Cell cytotoxicity was measured using an MTT assay. The autophagy pathway was studied by Beclin-1 immunoassay. Data were statistically analyzed between different transfected groups. It has been shown that the VSV M protein induced higher levels of Beclin-1 than the M51R mutant in the BT-20 cell line. Increased levels of Beclin-1 were also associated with VSV M cell-induced cytotoxicity. It has been shown here that VSV wild type or mutant M proteins can cause autophagy-induced cell death by increasing Beclin-1 expression. This includes the possible role of VSV to be used as an oncolytic virus in breast cancer treatment.  Different treatments have been used to defeat cancer cells. However, with the discovery of oncolytic features of certain viruses or oncolytic viruses (OVs), researchers have drawn attention to the use of these viruses to destroy cancer cells . OVs are multi-mechanistic antitumor agents that directly infect and destroy cancer cells . OVs cause oncolysis directly via apoptosis, necrosis, pyroptosis, and autophagy . Tumor cells with Defective Interferon Response and Defects in Ras, p53 or Myc signaling pathways have also been shown to be susceptible to VSV replication . There are a variety of recognized viruses with oncolytic abilities. One of these viruses, ONCORINE, is now approved for cancer treatment . Talimogene laherparepvec (T-VEC), a genetically modified herpes virus, was another viral drug used to treat advanced melanoma . More viruses, including Newcastle Disease Virus ( NDV), Parvoviruses, Myxoma virus, Reovirus, Seneca Valley Virus have an inherent oncolytic capability . Some genetically modified viruses such as Vesicular Stomatitis Virus (VSV) are also potent to be used for cancer oncotherapy. VSV is a fast-replication, negative-sense RNA virus. A natural wide range of VSVs and its high malignant cell tropism, rare pre-existing immunity, simple biological structure, and weak or no human pathogenicity are the main factors that make VSV an appropriate option for oncolytic cancer virotherapy . VSV comprises five proteins, namely nucleocapsid (N), matrix (M), phosphoprotein (P), glycoprotein (G), and broad polymerase (L) proteins . M protein plays a crucial function in the translation of viral proteins within infected host cells . However, M protein is known to direct the oncolytic process to the tumor site. Studies have shown that cancer cells are more prone to VSV containing the substituted M protein (M51R) . The mutant strain of VSV was thus introduced to be used in therapeutic approaches.Further studies indicate the safety of oncolytic use of VSV in the treatment of cancer cells. As potent inducers of interferon type 1 response, VSV R51M mutants are not capable of replicating within normal cells . The first analysis of M51R was based on a review of the temperature sensitive (ts) strain of VSV, Ts082, grown on the BHK-21 cell line. Coulon et al.’s research has shown that VSV containing M51R substitution is capable of shutting down the cellular RNA synthesis machine and for effective viral transcription . In another study by Hastie, et al, VSV-ΔM51 was shown to be capable of infecting pancreatic adenocarcinoma (PDA) cell lines in the mouse. This recombinant virus has been able to replicate and destroy cancer cells in Vivo . These data indicate that VSV M mutant can lead to cancer cells through any of the mechanisms behind inducible cell dead. Autophagy is one of the essential mechanisms by which VSV can cause cell death.becline-1 gene was found to be inhibited in 75% of ovarian, 50% of breast and 40% of prostate cancer . Beclin-1 expression has been reported to be different in various cell lines . NF-kB signaling is involved on the autophagy pathway. In a study by Shulak et al, NF-kB inhibition was found to inhibit VSV replication and subsequently inhibit cancer cell death . In primary chronic lymphocytic leukemia (CLL) cells after VSV infection, the mechanistic role of autophagy in mediating cell death has been shown . In addition, it was found that VSV induces apoptosis accompanied by autophagy through strong activation of caspase-3 . Overall, more evidence must be given for the role of VSV-induced autophagy in cancer-cell death.Autophagy is a homeostatic cell recycling process that helps to destroy damaged cellular proteins and organelles in all living cells . The role of autophagy in cancer is controversial . Becline-1 is a coiled-coil protein and an essential primary autophageal molecule . The The goal here was to investigate whether a construct expressing M51R mutant is capable of inducing cancer-cell death in the breast cancer cell line, BT-20, via autophagy. Cell culture and VSV preparation2 at 37 °C. The reagents were provided from the previous study . The medium was replaced two times per week with fresh complete medium. After reaching to >90% confluence, cells were harvested and seeded for transfection. A VSV was kindly provided by Dr. Hadi Razavi Nikoo. The virus with multiplicity of infection (MOI) 5 was used as a positive control for western blot and ELISA. BT-20 human breast cancer cell line was purchased from the Iranian Biological Resource Center . The cell line was propagated in tissue culture flasks with complete medium containing Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum , 100μg/ml streptomycin (Gibco), which atmosphere containing 5% COPlasmids and transfection4 cells/well were seeded onto 96-well plates containing complete medium. After confluence reached to >90%, 100 ng of each construct along with 2 µl of lipofectamine2000TM reagent were used for transfection. Briefly, the constructs and lipofectamine2000TM reagent were diluted with antibiotic-free DMEM-F12 separately and placed at room temperature for 10 minutes. Then constructs mixed with lipofection reagent at final volume of 50 μl. The mixture was stored at 37°C for 45 minutes. After 45 minutes, mixtures were added onto the cells. Cells were kept at 37°C with 5% CO2 for 4 hours. Ultimately, the transfection solution was aspirated and replaced with 100 μl of complete medium. 24 hours later cells were screened for transfection efficiency by fluorescence microscopy .VSV M wild type and R51M mutant construct (pcDNA3.1-wt and pcDNA3.1-M51R) provided from the previous study . After optimization, BT-20 was transfected with either pcDNA3.1-wt or pcDNA3.1-M51R lipofectamine2000TM reagent , 3 × 10Infectivity assay4 cells/well were seeded into a 96-well plate. Cells were exposed to eitherpcDNA3.1-wt, pcDNA3.1-M51R, or empty vector. For each construct, two concentrations (100 and 200 ng) were used. The cell viability was assessed by adding 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) to transfected wells after 24hr, 48hr, and 72hr post transfection. Each test was repeated for three time in triplicate. After 3 to 4 hours later, the supernatant was discarded and Formazan crystals were dissolved in Dimethyl sulfoxide (DMSO). Thirteen minutes later, the optical density (OD) was read at 570 nm.3 × 10Western blot analysis106 BT-20 cells were seeded into a 6-well plate. 24hr after propagation, cells were transfected with either pcDNA3.1-wt, pcDNA3.1-M51R, or pcDNA3.1 empty vector. Each test performed in duplicate. 24 hr later cells were lysed with lysis buffer . Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% polyacrylamide gels, then gels were electro-blotted onto a nitrocellulose membrane and blocked with 3% bovine serum albumin (BSA). The membrane was washed (PBS+Tween-20) 5 times. The membrane was then incubated with primary mouse anti-VSV M monoclonal antibody  at 4ºC overnight. Next day membrane washed as the same. Next, HRP-conjugated Goat-anti-mouse IgG secondary antibody was inoculated to the membrane. Bands were observed by using Electro Chemo-Luminescence .Human Beclin-1immuno assay4 BT-20 human breast cancer cells were grown in 24-well plate as explained above. Cells were transfected with either pcDNA3.1-wt or pcDNA3.1-M51R. Cells were lysed by sonication at 24 hr, 48 hr, and 72 hr post transfection. Experiments were performed in duplicate. Beclin-1 activity was determined by using Sandwich ELISA  according to the manufactured protocol (Life Span Biosciences). Briefly, standard sample was diluted and read at 450 nm to make a standard curve. Concentration of Beclin-1 in test or control samples was calculated according to the standard. Furthermore, a mock cell and VSV infected BT-20 were used as a negative and positive controls, respectively.3 × 10Statistical analysis softwareGraphs and statistics were performed using Microsoft office Excel and GraphPad Prims v7. For statistical analysis, Tuckey, t-test and ANOVA tests were used. The p-values of less than 0.05 were considered as statistically significant.Optimization of BT-20 breast cancer cell line transfection using lipofectamine 2000The efficient and successful transfection was achieved at 100ng/µl plasmid 2μl lipofectamine .MTT assay was performed to evaluate the cytotoxicity induced by VSV M wild type and VSV M M51R mutant. As it is illustrated in The expression of the VSV M protein was further evaluated by using western blot. In this assay, BT-20 was infected with VSV virus at 5 moi. A quantitative sandwich ELISA with specificity ranging from 0.156 ng/ml to 10 ng/ml was used for quantification of Beclin-1. A standard curved has been constructed (R2=0.99) according to the provided instruction. Breast cancer is one of the most common cancers among women in the world and is still on the grow . There are few common treatments against breast cancer, including surgery, chemotherapy (Greco and Marotti 2006), radiotherapy , immune therapy , and targeted therapy . Despite a number of therapies, it has been shown that they have not been effective and that there are deaths among patients every year. New therapeutic approaches must also be tested. Oncolytic virotherapy using non-human viruses is a new therapeutic strategy used for cancer treatment. Various studies have focused on the therapeutic use of Vesicular Stomatitis Virus (VSV) in various cancers. Thus, it has been shown that VSV is effective in the treatment of cancers, such as endometrial cancer , malignant melanoma , pancreatic adenocarcinoma , glioblastoma multiform , neuroendocrine tumor , Head and neck squamous carcinoma , hepatocellular carcinoma , prostate cancer , hematologic malignancies , and colorectal cancer . The effectiveness of this virus in the treatment of breast cancer was also tested on different cell lines of the breast cancer . It has been shown that deletion of methionine 51 in the VSV matrix protein improves its tumor specificity and prevents its involvement in normal cells with healthy immune protection . Therefore, we investigated in this study the ability of constructs expressing VSV wild type and VSV matrix protein mutant M51R to induce cancer cell death by triggering the autophagy pathway. The toxicity was measured at different time points by MTT in the BT-20 cell line transfected with 100 ng and 200 ng pcDNA3.1-wt or pcDNA3.1-M51R. The findings showed that at 12 hr, 48 hr, and 72 hr post transfection, two proteins had high toxicities (>70%) at all concentrations. These results were consistent with previous studies, which demonstrated the toxicity of both mutant and wild M proteins in the cancer cell lines in concern . The mutant M protein has been shown to have more propensity to interferon-deficient cancer cells than the wild type . Interestingly, the toxicity caused by wild type M protein at two concentrations at any given time was significantly higher than that caused by M51R mutant. This means that of VSV M51R mutant can be used for oncolytic virotherapy in BT-20 cell line. Since both concentrations had the same effects, further assays were performed using 100 ng/μl. Whether or not cell death occurred because of the autophagy pathway, the level of Beclin-1 analyzed in the transfected cells. At 24 hours after transfection the expression of Beclin-1 was undetectable in the transfected cells. In the mean time, Beclin-1 was overexpressed in the VSV inoculated cell line. This was partly due to either plasmids or lipofection reagent-induced cell death. The Beclin-1 expression, however, was restored at 48hr and 72hr post transfection. At 48 hr an increased expression was observed in Beclin-1 and reached its peak at 72 hr post-transfection. The amount of Beclin-1 was, however, decreased at the next two points in time. This is due to cell death caused in pathways other than autophagy by the virus .These results are also consistent with other studies. Nguyen et al, demonstrated that induction of the Nuclear factor erythroid 2-related factor 2 (Nrf2) signal is needed to replicate VSVD51 and oncolysis in both in vitro and in vivo . In another study, Shulak et al, showed NF- κ B activity through RELA/p65 signaling modulation, causing autophagy induction, enhancing VSV replication, and eventually causing cancer cells to die .Beclin-1 gene region have mono-allelic deletion . The present study provides further evidence of the significance of autophagy and Beclin-1 to cause cell death in the breast cancer cell line BT-20.Autophagy is a very complex process. The role of autophagy in cancer therapy is controversial, despite the numerous studies performed in this area. Dependent on the stage of cancer, autophagy seems to be geared towards a prophylactic approach to tumor suppression . There is evidence that autophagy performs a tumor suppressor through its regulatory Beclin-1 protein, which has an anti-tumor role . Previously, the significance of autophagy and particularly of Beclin-1 activity has been shown . It has been shown that 40-75% of sporadic human breast cancers in the Here, BT-20 was used as a breast cancer cell line. It should be noted that the cell line is very slow growth (63 hr duplication time) and chemotherapy-resistance. However, for the first time we report the autophagy induced with VSV and its M51R mutant and wild type M proteins in that cell. The Beclin-1 concentrations were evaluated in this analysis. However, evaluation of the other approaches leading to an increase in autophagy is warranted .In conclusion, here, both wild type VSV and mutant M proteins have been shown to be able to kill breast cancer cells via the autophagy pathway. This suggests the possible role of the VSV M protein in the treatment of breast cancer for use as an oncolytic virus. With the evidence given here, VSV M proteins can be used to induce cell death in the chemotherapy-resistant cell lines.


We designed this prospective observational study to clarify the associations between dialysate IL-6, a marker of ongoing peritoneal inflammation, Tie2, an important factor in angiogenesis in the peritoneum, and a high peritoneal solute transport rate (PSTR) in patients undergoing peritoneal dialysis (PD) and to investigate their outcome predictive roles.A total of 60 stable continuous ambulatory peritoneal dialysis (CAPD) patients from a single center in China were analyzed in this prospective study. We measured dialysate levels of IL-6 and Tie-2 using ELISAs. Our primary study endpoint was all-cause mortality with 10 years’ follow-up.For the evaluation of PSTR, we used the Dialysis/Plasma creatinine (D/Pcr) ratio. We subdivided the patients into two groups for statistical evaluation: low and low average D/Pcr , and high and high average D/Pcr  transporters. The mean levels of dialysates IL-6 (21.71 ± 8.88 pg/mL) and Tie-2 (1.23 ± 0.43 ng/mL) were significantly higher in the H/A . Moreover, IL-6 and Tie-2 were positively correlated with D/Pcr . Both dialysates IL-6 and Tie-2 were independent determinants of a high peritoneal solute transport rate. After follow-up for 42.65±18.08 months, 30 patients (50.0%) had died. An increased D/Pcr increased the risk of all-cause mortality in patients with CAPD (p=0.018), but the dialysates IL-6 and Tie2 were not independent predictors of all-cause mortality (p>0.05).Our results suggest that patients undergoing CAPD have a high peritoneal solute transport status with local peritoneal inflammation and angiogenesis. Increased D/Pcr is a relative risk factor for mortality and technique failure in patients undergoing CAPD. Peritoneal dialysis (PD) is a well-established renal replacement therapy for patients with end-stage renal disease (ESRD).9Interleukin-6 (IL-6) is a pleiotropic cytokine with roles in multiple pathological and physiological processes and a central regulator of inflammatory processes. Local intraperitoneal IL-6 production is a sign of an intraperitoneal inflammatory state.10Tie-2, a transmembrane tyrosine kinase receptor for Ang ligands crucial for angiogenesis, blood vessel maturation, and vascular endothelium integrity.12We hypothesized that intraperitoneal inflammation and angiogenesis are interrelated and associated with a high peritoneal solute transport status in patients undergoing PD. In this study, we analyzed the intraperitoneal IL-6 and Tie-2 levels in 60 patients undergoing stable CAPD treatment with glucose-based solutions. Moreover, we assessed the association between intraperitoneal IL-6 and Tie-2, and their possible correlation to the PSTR during stable CAPDs.We performed this prospective observational study with data from a single center in China. We recruited all the study participants between July and December of 2007 and prospectively followed them to the end of the study 10 years later . All procedures followed in this study were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975. The Human Research Ethics Committee of Renji Hospital, School of Medicine, Shanghai Jiao Tong University approved the study protocol . All participants gave their written informed consent.The 60 patients on CAPD represented all of the clinically stable patients treated in the center and were regularly followed up in the Dialysis Unit of Renji Hospital affiliated to Shanghai Jiao Tong University School of Medicine. This population consisted of 29 men and 31 women with a mean age of 57.9±13.9 years , treated with CAPD for an average of 34.2±29.1 months (3–149.6 months). ®, Baxter, USA) and recorded its exact time.On the night before the first dialysis fluid drain for the study, all the patients underwent a dialysis exchange using a 2.5% glucose solution . Both of the assays are considered highly specific for the cytokines, and we observed no significant cross-reactivity. The plates were read using a microplate reader .We determined the concentrations of cytokines in the dialysis effluent after the night dwell using the ELISA technique. All samples were run simultaneously and in duplicate for each mediator to avoid intra- and inter-assay variations. IL-6 and Tie-2 levels were determined using QuantikineWe followed up all the patients prospectively from the enrollment of the study until death, cessation of PD, transfer to other centers, or the end of the study . We evaluated death from all causes as the primary outcome, and limited the survival analysis to the first event.t-tests. When normal distribution was not present, we applied a non-parametric analysis (Mann–Whitney test). To compare D/Pcr values with their respective cytokine levels, we applied a linear regression analysis. We generated cumulative survival curves using the Kaplan–Meier method. We also applied the Cox proportional hazards model to estimate the relative risks of all-cause mortality for different variables. We performed all statistical tests using the SPSS software version 25.0 for Windows .We expressed results as mean values ± standard deviations (SDs), and used medians and ranges for non-normally distributed parameters. To compare differences between two transport groups, we used unpaired Student The characteristics of the two transport groups are described in The concentration of IL-6 in the dialysate was significantly higher in the H/A group than in the L/A group (p=0.001) . The diaMeanwhile, the concentration of Tie-2 in dialysate was significantly higher in the H/A group than in the L/A group (p=0.019) . The diaWe carried out a multivariate linear regression analysis with age, dialysis age, BMI (body mass index), blood glucose, albumin, CRP, dialysate concentration of IL-6, and the dialysate concentration of Tie-2 as the independent variables, and with the D/Pcr value as the dependent variable. Our results show that the dialysate concentrations of IL-6  and Tie-2  were both independent risk factors for D/Pcr .After a mean follow-up of 42.65±18.08 months, 30 patients (50.0%) had died and 21 (35.0%) had 21 continued CAPD; two patients (3.3%) had undergone kidney transplantation, 6 (10.0%) had been switched to permanent hemodialysis therapy, and one patient (1.7%) had transferred to another center. According to the Kaplan–Meier survival analysis, the all-cause mortality was significantly higher in the L/A group than in the H/A group p=0.018; , while oThe patients on PD and with high peritoneal permeability had an increased small-solute PSTR; the peritoneal osmotic pressure gradient disappeared rapidly, resulting in a reduced fluid clearance rate. A high PSTR reflects the large effective peritoneal surface area resulting from a large number of perfused peritoneal capillaries engaged in the diffusion of the solute, and the increased permeability of the peritoneal vasculature. The most common functional alteration during long-term CAPD is an increased peritoneal small-solute transport rate.9We found that patients with higher PSTR had higher levels of dialysate IL-6 and Tie-2, indicating these are closely associated with a small-solute PSTR. Moreover, we found that dialysate concentrations of IL-6 and Tie-2 were independent risk factors of D/Pcr. These results suggest a link between inflammation, angiogenesis, and the small-solute PSTR. However, we found no association between dialysate concentrations of IL-6 and Tie-2.The acute phase inflammatory response factor IL-6 is secreted by different cells in response to diverse stimuli, it induces the production of acute phase proteins and regulates the temporal switch from acute to chronic inflammation.21In this study, the level of dialysate IL-6 was significantly higher in the H/A group than in the L/A group. Moreover, the dialysate concentration of IL-6 had a positive correlation with the D/Pcr. Consistent with the results of a previous study, ours also suggested a close link between a high PSTR and local peritoneal inflammation, and we found that the high peritoneal transport patients presented a more severe local intraperitoneal inflammation state than the others.IL-6 has been detected in the dialysate of patients lacking signs of systemic inflammation.25The principal water and solute barriers in PD are the blood capillary endothelium and the interstitial matrix. The peritoneal permeability increases in patients undergoing PD and, eventually, leads to ultrafiltration failure. Peritoneal biopsies in patients undergoing PD have shown that extensive angiogenesis is associated with peritoneal treatment duration.Interestingly, we also found that the dialysate Tie-2 level was positively correlated with the blood glucose level. Glucose is a proinflammatory agent with additional profibrotic effects. A high concentration of glucose in the dialysate may promote angiogenesis and fibrosis of the peritoneal membrane through a stimulation of the polyol pathway.Studies have demonstrated an intimate connection between a high transport status and poor outcomes.First of all, we only performed single-time-point measurements of dialysate concentrations of IL-6 and Tie-2 that may not reflect changes over time or time-averaged exposures. Second, the number of cases included in this study is relatively small. another well-designed and extensive study will be required to confirm our findings.In summary, our results suggest that patients undergoing CAPD have a high PSTR with local peritoneal inflammation and angiogenesis. The dialysate concentrations of IL-6 and Tie-2 both seem to be independent risk factors for peritoneal permeability. In our patients, peritoneal local inflammation and angiogenesis were both involved in the peritoneal permeability changes observed. Increased D/Pcr was a relative risk for mortality and technique failure in these patients.YH and WF conceived, designed the study and are responsible for integrity of the study.HZ and ZL collected the data.HY performed the analysis.YH was involved in the writing of the manuscript.All authors have read and approved the final manuscript.


We screened four databases  for the observational studies about the OSA and T2DM. Studies were collected from database establishment to October 2020. We performed a traditional subgroup meta-analysis. What is more, linear and spline dose-response models were applied to assess the association between apnea-hypopnea index (AHI), an indicator to evaluate the severity of OSA, and the risk of T2DM. Review Manager, version 5.3, software and Stata 16.0 were used for the analysis. P < 0.05). Moderate-dose AHI increased the risk of T2DM with a higher odds ratio . Moderate-to-severe-dose AHI increased the risk of T2DM with a higher odds ratio . Severe-dose AHI increased the risk of T2DM with a higher odds ratio . Furthermore, the spline and linear dose-response meta-analysis results revealed that the risk of T2DM increased with increasing AHI values.  Seven observational studies were included in the research. We excluded a study in the conventional meta-analysis. In the subgroup analysis, mild-dose AHI increased the risk of T2DM (odds ratio = 1.23, 95% confidence interval = 1.06–1.41,  Through the dose-response meta-analysis, we found a potential dose-response relationship existed between the severity of OSA and the risk of T2DM. This relationship in our passage should be considered in the prevention of T2DM in the future. Obstructive sleep apnea (OSA) is one of the sleep disorders due to the refractory hypoxemia episodes and sleep fragments, leading to daytime sleepiness, impaired performance, and reduced quality of life.b=rSy/Sx to estimate the corresponding AHI [There are 90% elderly males and 78% elderly females suffering from OSA approximately worldwide . The apnding AHI . Intermiding AHI , suggestType 2 diabetes mellitus (T2DM) is a systemic disease and a massive health threat, causing considerable damage to each organ. The incidence of T2DM is still rising, which has become severe and expanding worldwide wellbeing burden. The global figures read that 381.8 million adults are affected, and these data will come to 591.9 million in 2035. In developed countries, most T2DM patients are older than 50, while in developing countries, 41% of T2DM are elderly [According to previous research works, OSA was closely associated with the high death rate from all causes. OSA damages all the systems in human bodies and organs with significant vascular structure, including the brain, heart, and kidneys. Besides, Xu et al.  reportedOSA has been defined as a risk factor for T2DM by many studies . MoreoveIn our present research, there were seven observational studies included. We planned to explore the association between OSA and different T2DM severity from these six OSA-related studies. To ensure our results' preciseness, we not only did a traditional meta-analysis but a dose-response meta-analysis was also accomplished. This study would offer strong evidence and new thought for the prevention of T2DM for OSA patients.We registered this systematic review and dose-response meta-analysis with the INPLASY register (INPLASY2020110027). In addition, we followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines for meta-analyses.In the present study, we included cross-sectional studies, cohort studies, and case-control studies that had clear outcomes and reported hazard ratio (HR), odds ratio (OR), or relative risk (RR) and 95% confidence interval (CI) for the association between OSA and T2DM. HR and OR were considered approximately RRs . The resFurthermore, the participants' ages were older than 18 years. We excluded patients with type 1 DM, and researchers used other indexes as indicators. As for the research type, case reports and reviews were also excluded. The criteria for inclusion and exclusion are shown in We screened the PubMed, Embase, Cochrane Library, and CNKI (Chinese) databases. The retrieval time was from incipiency to September 2020. To fit different demands in different databases, we modified the search terms and strategy. In addition, we screened all references of the included articles to ensure we collected the related studies as many as possible. Moreover, we communicated with senior specialists when conceivable to complete the search methodology. The search strategy in PubMed is shown in Supplementary Materials Two independent investigators (YZX and JXC) extracted data from the included articles. Discrepancies were handled by consultation and guide from JQH. In addition, data about baseline information of participants, study design, and relevant statistics were extracted. No qualification was made to measure the seriousness stratification of T2DM.We evaluated the included studies according to the Newcastle-Ottawa scale (NOS) . A quantTwo investigators (YZX and YF) finished the conventional meta-analyses with Cochrane Review Manager, version 5.3, software to assess a specific outcome's risk.Q and I2. We adopt a standard for P-value that P value < 0.1 means the results possessed statistical heterogeneity. I2 describes the extent of variation due to heterogeneity rather than chance. The lower the I2 is, the less the variation is. I2 < 25% was considered little heterogeneity; 25% < I2 < 50%, a little heterogeneity. I2 > 50% showed there existed enough heterogeneity to select a random-effects model. While I2 < 50%, a fixed-effect model was employed [The evaluation of heterogeneity among studies was carried out with the use of employed .P < 0.05 means the existence of a significant difference.Funnel plots were selected to assess whether the report existed publication bias. Egger's and Begg's texts were designed to recognize the plots' asymmetry, suggestive of bias. In this plot, α = 0.05 was taken as a cut-off for the regression parameters [b = r×sy/sx [For further research, we performed a dose-response meta-analysis using Stata, version 16.0, software. We performed the dose-response meta-analysis based on a two-step method , 16. Firrameters . For a s r×sy/sx . Accordi r×sy/sx , the difThis study complied with the Declaration of Helsinki. Given the study was a meta-analysis, no prior ethical approval was required.After a primary search, we identified 537 articles. There were 49 duplicated publications. In the left passages, from the title, we knew that there were 160 reviews. After abstracts screening, we excluded 290 studies, for they were not related to the topic. Then, we screened the whole text. We found 15 passages that did not mention the indicators we were interested in, and ten articles did not declare accurate RR/OR or 95% CI. Five research works did not perform OSA subgrouping. Natalia et al.  was a leAll the research studies included in this meta-analysis were cohort studies, including 15252 participants. The study sizes were different from each other (303 to 8678). There existed a sex difference between the included research studies (women count 0 to 54.2%). As for the outcomes of the research works, 2381 new cases of T2DM were diagnosed . The preP = 0.002) (P < 0.001) for moderate (P < 0.001) for moderate-to-severe (P < 0.001) for severe (All the included studies researched the correlation between mild-dose AHI (5–15) and the risk of occurrence of T2DM. Three observational studies paid attention to the effect of moderate-to-severe-dose AHI (>15). Only two studies reported the association between moderate-dose AHI (15–29) and T2DM. Moreover, some research did not select 0 < AHI < 4.9 as a reference, which led to difficulty in merging these data. Besides, Appleton et al.  only per= 0.002)  for mildmoderate , OR = 2.o-severe , and OR r severe .P < 0.05 means that there existed bias in the selection of the studies. The P value of Begg's or Egger's texts for all subgroups was more than 0.05. The results of the heterogeneity in the different subgroups were as follows: 0 (P = 0.47) for mild, 59% (P = 0.12) for moderate, 0 (P = 0.8) for moderate-to-severe, and 79% (P = 0.008) for severe. For further heterogeneity research, the funnel plot , which means the existence of a linear model. We performed linear (P < 0.05). The spline analysis showed that the risk of occurrence of T2DM increased with increasing OSA severity.There were statistically significant differences in the risk of T2DM between the mild-dose, moderate-to-severe-dose, and zero-dose groups of OSA. What is more, the linear model test result was not significant . This suggested the result of the conventional meta-analysis was receivable, but accepting should be cautious. We speculated heterogeneity might be for the following reasons. First, there were different proportions of age and gender participants in the included studies. Male OSA has higher AHI compared to age-matched females [For the meta-analysis of two subgroups, the mild-dose subgroup and moderate-to-server-dose AHI analysis showed heterogeneity when merging the related research works. The mild-dose, moderate-dose, and moderate-to-severe-dose subgroups' heterogeneity showed no significant differences; the severe-dose subgroups were gross ( females . Older a females . The pre females . Second, females . Reichmu females  selectedOur study provided strong evidence that OSA was related to the risk of T2DM occurrence and performed a prediction for the RR to different AHI doses with the linear and spline models. The results were consistent with previous meta-analyses –30. In QOSA is characterized by intractable hypoxemia, which leads to various pathologic conditions, including neural activation, systemic inflammation, oxidative stress loading, and hormone disorder. The changes in hormonal systems will add an influence on energy metabolism. Xu et al.  and ZhanThere are a few impediments to our research. First, some studies' sample size was limited. Marshall et al.  only resOur research has proved that OSA is a risk factor for DM. Besides, for different severity, OSA shows a different OR. As a quantitative indicator of OSA, the AHI was positively related to the risk of DM, which indicates that doctors should pay more attention to the patients' breath events during sleep after ablation treatment. In this research, we set up a dose-response model for AHI and DM. With the help of the model we built, physicians will have more evidence to decide the intervention time for OSA patients to prevent DM.In a word, our study concluded that the AHI is positively correlated to the risk of DM occurrence. OSA is associated with the occurrence of T2DM. Further studies are needed to assess whether OSA treatment would decrease T2DM risk and benefit the management of T2DM.